Development and application of a fast, reproducible assay to measure HCV NS3 protease activity using Escherichia coli lysate.

The hepatitis C virus (HCV) NS3/4A protease is a key target of efforts to develop direct-acting antiviral inhibitors for treatment of chronic HCV infection. In vitro analyses of the effects of NS3/4A mutations and polymorphisms on protease inhibitor (PI) susceptibility are essential to nonclinical and clinical compound characterization, but can be hampered by time and technical limitations of current in vitro methods using replicon or purified protein systems. We have developed a fast and simple method utilizing full-length NS3/4A protease inducibly expressed in Escherichia coli cells. Minimally processed E. coli whole cell lysate was used for analyzing NS3/4A protease activity and inhibition by antiviral compounds. Assay conditions were optimized to develop a reproducible assay that can be used for efficient analysis of NS3 protease mutants with poor replication capacity in the replicon system. IC50 fold-changes for NS3 mutants relative to their wild-types generated by this NS3 assay are comparable to those observed in the replicon system, with an R(2) of 0.82 for the values obtained by the two methods. In addition, we demonstrate that this assay can be successfully used for population and clonal phenotyping of patient samples and characterization of PIs against the NS3/4A protease from HCV genotypes 1-6.

Synthesis of 2'-O,4'-C-alkylene-bridged ribonucleosides and their evaluation as inhibitors of HCV NS5B polymerase.

The synthesis of 2'-O,4'-C-methylene-bridged bicyclic guanine ribonucleosides bearing 2'-C-methyl or 5'-C-methyl modifications is described. Key to the successful installation of the methyl functionality in both cases was the use of a one-pot oxidation-Grignard procedure to avoid formation of the respective unreactive hydrates prior to alkylation. The 2'-C-methyl- and 5'-C-methyl-modified bicyclic guanosines were evaluated, along with the known uracil-, cytosine-, adenine-, guanine-LNA and guanine-ENA nucleosides, as potential antiviral agents and found to be inactive in the hepatitis C virus (HCV) cell-based replicon assay. Examination of the corresponding nucleoside triphosphates, however, against the purified HCV NS5B polymerase indicated that LNA-G and 2'-C-methyl-LNA-G are potent inhibitors of both 1b wild type and S282T mutant enzymes in vitro. Activity was further demonstrated for the LNA-G-triphosphate against HCV NS5B polymerase genotypes 1a, 2a, 3a and 4a. A phosphorylation by-pass prodrug strategy may be required to promote anti-HCV activity in the replicon assay.

A randomized, double-blind, multiple-dose study of the pan-genotypic NS5A inhibitor samatasvir in patients infected with hepatitis C virus genotype 1, 2, 3 or 4.

BACKGROUND & AIMS: Samatasvir is a pan-genotypic inhibitor of the hepatitis C (HCV) non-structural protein 5A (NS5A). This study evaluated the antiviral activity, pharmacokinetics and safety of samatasvir monotherapy in treatment-naïve subjects infected with HCV genotype 1-4. METHODS: Thirty-four genotype 1 and thirty genotype 2, 3 or 4 subjects were randomized to receive for 3days placebo or samatasvir 25-100mg per day. Plasma samples for HCV RNA, pharmacokinetics and sequencing were collected up to day 10. RESULTS: Samatasvir achieved potent antiviral activity across genotypes: mean maximum reductions from baseline were 3.2-3.6 (genotype 1a), 3.0-4.3 (genotype 1b), 3.2-3.4 (genotype 3), and 3.6-3.9 (genotype 4) log10/ml respectively; no viral rebound was observed during the 3-day treatment period. For genotype 2 HCV, samatasvir was active in subjects with NS5A L31 polymorphism at baseline (individual range 2.5-4.1 log10/ml), but showed minimal activity in those with baseline M31 polymorphism. Samatasvir exhibited a long plasma half-life of approximately 20h which supports once daily dosing. Samatasvir was well tolerated in all subjects with no safety-related discontinuations or serious adverse events. The most common adverse events included constipation, nausea and headache and occurred at similar frequency in active and placebo subjects. All events were mild or moderate in intensity. There were no patterns or dose dependence of adverse events, vital signs, laboratory parameters or electrocardiograms. CONCLUSIONS: Samatasvir 25-100mg monotherapy for 3days was well tolerated and induced a rapid and profound reduction in plasma HCV RNA in subjects infected with HCV genotype 1-4. Samatasvir is being evaluated in combination with other direct-acting antiviral agents in subjects with HCV infection.

Rapid decline of viral RNA in chronic hepatitis C patients treated once daily with IDX320: a novel macrocyclic HCV protease inhibitor.

BACKGROUND: The addition of direct-acting antivirals to pegylated interferon-α plus ribavirin for the treatment of chronic HCV infection can result in an increased sustained viral response rate and may permit reduction in treatment duration. IDX320 is a potent non-covalent macrocyclic inhibitor of the HCV NS3/4A protease. METHODS: This was a randomized double-blind placebo-controlled single- and multiple-dose study to assess the safety, tolerability, antiviral activity and pharmacokinetics of IDX320 in healthy volunteers (HV) and patients with chronic HCV genotype 1 infection. HV (n=48) received single or multiple ascending doses of IDX320. Two HCV-infected patients received a single dose of 200 mg IDX320. Dosages for other HCV-infected patients were as follows: placebo, 50, 100, 200 or 400 mg of IDX320 orally once daily for 3 days (n=30) or placebo/200 mg of IDX320 twice-daily for 3 days (n=8). RESULTS: In total, 48 HV and 40 HCV-infected patients were enrolled and all completed the study. There were no serious adverse events. The majority of adverse events were of mild or moderate intensity. Pharmacokinetics supported a once-daily dosing regimen. A rapid decline in plasma HCV RNA was observed in all patients. In the multiple-dose study, mean HCV RNA reductions were 2.6, 3.1, 3.1, 3.3 and 3.8 log(10) IU/ml after 3 days in the IDX320 50, 100, 200, 400 mg once-daily and 200 mg twice-daily treatment groups, respectively. This compared to a mean HCV RNA reduction of 0.04 log(10) in the placebo group. CONCLUSIONS: Once-daily IDX320 dosing demonstrated potent dose-dependent antiviral activity in treatment-naive HCV genotype-1-infected patients.

Evidence that the human cytomegalovirus IE2-86 protein binds mdm2 and facilitates mdm2 degradation.

Levels of the p53 tumor suppressor protein are increased in human cytomegalovirus (HCMV)-infected cells and may be important for HCMV pathogenesis. In normal cells p53 levels are kept low due to an autoregulatory feedback loop where p53 activates the transcription of mdm2 and mdm2 binds and ubiquitinates p53, targeting p53 for proteasomal degradation. Here we report that, in contrast to uninfected cells, mdm2 was undetectable upon treatment of infected fibroblasts with the proteasome inhibitor MG132. Cellular depletion of mdm2 was reproducible in p53-null cells transfected with the HCMV IE2-86 protein, but not with IE172, independently of the endogenous mdm2 promoter. IE2-86 also prevented the emergence of presumably ubiquitinated species of p53. The regions of IE2-86 important for mdm2 depletion were those containing the sequences corresponding to the putative zinc finger and C-terminal acidic motifs. mdm2 and IE2-86 coimmunoprecipitated in transfected and infected cell lysates and in a cell-free system. IE2-86 blocked mdm2's p53-independent transactivation of the cyclin A promoter in transient-transfection experiments. Pulse-chase experiments revealed that IE2-86 but not IE1-72 or several loss-of-function IE2-86 mutants increased the half-life of p53 and reduced the half-life of mdm2. Short interfering RNA-mediated depletion of IE2-86 restored the ability of HCMV-infected cells to accumulate mdm2 in response to proteasome inhibition. Taken together, the data suggest that specific interactions between IE2-86 and mdm2 cause proteasome-independent degradation of mdm2 and that this may be important for the accumulation of p53 in HCMV-infected cells.

A role for Saccharomyces cerevisiae Cul8 ubiquitin ligase in proper anaphase progression.

We have undertaken a study of the yeast cullin family members Cul3 and Cul8, as little is known about their biochemical and physiological functions. We demonstrate that these cullins are associated in vivo with ubiquitin ligase activity. We show that Cul3 and Cul8 are functionally distinct from Cdc53 and do not interact with ySkp1, suggesting that they target substrates by Skp1- and possibly F-box protein-independent mechanisms. Whereas null mutants of CUL3 appear normal, yeast cells lacking CUL8 have a slower growth rate and are delayed in their progress through anaphase. The anaphase delay phenotype can be complemented by ectopic expression of Cul8 but not by any other yeast or human cullins, nor by a cul8 mutant deficient in binding to RING finger protein Roc1. Deletion of the RAD9 gene suppressed the anaphase delay phenotype of cul8delta, suggesting that loss of Cul8 function may compromise genomic integrity. These results indicate that in addition to the anaphase promoting complex, mitotic progression may involve another E3 ubiquitin ligase mediated by Cul8 protein.